The species of the genus Nannochloropsis belong to the Division Eustigmatophyta, Class Eustigmatophyceae, Order Eustigmatales, Family Monodopsidaceae (Hibberd, 1981, Bot J Linnean Society 82:93-119). The class was separated from the Xanthophyceae on the basis of its structure and cytology, and, subsequently, due to its pigment composition, since it lacks chlorophyll b (Hibberd & Leedale, 1972, Annals of Botany 36:49-71; Whittle & Casselton, 1975, British Phycological Journal 10:179-191).
Nannochloropsis cells are coccoid cells, with an approximate diameter of 2-4 μm, they do not have flagella and do not present moving states. They have a green-yellow colour, and for this reason may be confused with Chlorophyta cells (Santos, 1996, Beiheft Nova Hedwigia 112:391-405), which has led various publications to designate the Nannochloropsis species as marine Chlorella (Maruyama et al., 1986, Jap. J. Phycol. 34:319-325; Watanabe et al., 1983, Aquaculture 34:115-143). Apart from the morphological differences and the differences in pigment composition, since Chlorophyta present chlorophylls a and b, whereas Eustigmatophyceae only present chlorophyll a, in addition to various carotenoids, which may be used as a taxonomic character (Jeffrey & Vesk, 1997, Phytoplankton pigments in oceanography, S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright (Eds). UNESCO Publishing Paris, pp 37-84; Lubian & Establier, 1982, Investigación Pesquera 46:379-389), both groups present significant differences in their fatty acid profile, since, whereas chlorophytes do not contain fatty acids with more than 18 carbon atoms, species of the genus Nannochloropsis present a high percentage of the omega-3 polyunsaturated fatty acid eicosapentaenoic (20:5 n-3, EPA) (Ferreira et al., 2009, March Biotechnol 11:585-595; Sukenik et al., 1993, Cohen, Z. (Ed.), Chemicals from Microalgae. Taylor and Francis, London, p 41-56), which may represent up to 25% of the fatty acids in this group and which, in addition to being essential for application in aquaculture, has various functional properties in animals and humans (Siriwardhana et al., 2012, Se-Kwon Kim, (Eds), Advances in Food and Nutrition Research, Academic Press, 2012, Volume 65, Pages 211-222); this makes this genus important from the biotechnological and pharmacological standpoints. Most of the species described belong to marine or brackish habitats, and the first freshwater species, Nannochloropsis limnetica, was described in 2000 (Krienitz et al., 2000, Phycologia 39:219-227). The fatty acid composition of N. limnetica is similar to that of the marine species, with an EPA content that may be as high as 24% of the total fatty acid content (Freire et al., 2013, Aquaculture Conference 2013: Celebrating 40 Years of Aquaculture—November, 2013, Gran Canaria (Spain); Krienitz et al., 2006, Phycologia 39:219-227).
Various marine species of the genus Nannochloropsis are cultured all over the world, to be used in the live food chain for the larval culture of marine fish, and are amongst the most commonly used species in mariculture. The main application of the marine species of the genus Nannochloropsis is in the culture of rotifers of the genus Brachionus, which are used as live food for marine fish larvae. Rotifer culture is a process that requires high quantities of microalgae, since these represent the only diet that allows for sustained, stable production under continuous culture, with high densities (Yoshimura et al., 2003, Aquaculture 227:165-172; Bentley et al., 2008, J World Aquac Soc 39:625-635). Moreover, microalgae of the genus Nannochloropsis result in a better growth and biochemical composition of the rotifers than the yeast Saccharomyces cerevisiae, which may also be used as food for rotifers (Luzbens et al., 1995, Aquaculture 133:295-309), or other artificial diets (Aragão et al., 2004, Aquaculture 234:429-445; Srivastava et al., 2006, Aquaculture 254:534-543; Koiso et al., 2009, Nippon Suisan Gakk 75:828-833). The high content of long-chain polyunsaturated fatty acids (PUFAs), especially EPA, in the species of the genus Nannochloropsis has been identified as the reason for its high nutritional value in aquaculture, both in the case of the rotifer Brachionus plicatilis fed with marine species of this genus (Watanabe et al., 1983, Aquaculture 34:115-143), and in the case of the zebra mussel (Dreissena polymorpha), the water flea Daphnia magna or the freshwater clam Corbicula fluminea fed with the freshwater species Nannochloropsis limnetica (Wacker & von Elert, 2003, Oecologia 135:332-338; Wacker et al., 2002, Limnol. Oceanogr. 47:1242-1248; Basen et al., 2012, Oecologia 170:57-64; Wacker & Martin-Creuzburg, 2007, Functional Ecology 21:738-747). Recently, it has been disclosed that the freshwater species Nannochloropsis limnetica may be used in the culture of Brachionus plicatilis in sea water with excellent results (Freire et al., 2013, Aquaculture Conference 2013: Celebrating 40 Years of Aquaculture—November, 2013, Gran Canaria (Spain)).
Concentrates of the freshwater microalga Chlorella are also being successfully used to maintain dense cultures of the rotifer Brachionus sp., although the marine species of the genus Nannochloropsis produce similar or higher growth rates (Hirayama & Nakamura, 1976, Aquaculture 8:301-307; Maruyama et al., 1997; Kobayashi et al., 2008). Since the Chlorella biomass industrially produced under mixotrophic or heterotrophic conditions is deficient in vitamin B12, which is essential for the growth of rotifers, the commercial products of this freshwater microalga designed to be used in aquaculture are enriched with this vitamin, which, in general, is directly added to the culture medium (Maruyama et al., 1989, Nippon Suisan Gakkaishi 55:1785-1790; Maruyama & Hirayama, 1993, Journal of the World Aquaculture Society 24:194-198). In the case of the marine species of Nannochloropsis, which are autotrophically cultured, the addition of this vitamin to the culture medium or subsequent enrichment with it are not necessary to obtain maximum rotifer growth rates. Moreover, the main advantage of Nannochloropsis species over other species of unicellular algae, more specifically, over species of Chlorella, is their high Eicosapentaenoic acid (EPA, 20:5 (n-3)) content, absent in species of the genus Chlorella, which is essential for the development of the fish larvae and is transferred to them through the rotifers. Currently, there are various commercial refrigerated, frozen, condensed or lyophilised products based on marine species of Nannochloropsis which produce good results for rotifer growth (Luzbens et al., 1995, Aquaculture 133:295-309; Navarro et al., 2001, Hydrobiologia 452:69-77). These products compete with a commercial product called Chlorella SV-12 (Pacific Trading Co., Ltd., Chlorella Industry Co., Ltd. http://www.pacific-trading.co.jp/en/product/01-2.html), which is a Chlorella concentrate (approx. 13.5% dry weight) artificially enriched to contain 17% of the long-chain fatty acid docosahexaenoic acid (22:6 (n-3), DHA). As reported by the attached technical specifications of the product, this biomass contains only 2% EPA (EPA:DHA ratio, 1:8.5).
The process for enriching Chlorella with DHA has been previously disclosed for use in the culture of rotifers (Hayashi et al., 2001, Biosci. Biotechnol. Biochem. 65:202-204). Chlorella cells were heterotrophically cultured with glucose, and tuna oil (0.5%) containing 26.8% DHA or free fatty acids obtained from the hydrolysate of the same oil was added for 24 hours. These authors were not able to obtain enrichment using oils, and it was only possible to enrich different species of Chlorella by using free fatty acids, to 16.9% of the total fatty acid content (Hayashi et al., 2001, Biosci. Biotechnol. Biochem. 65:202-204), as the use of non-hydrolysed oils was not effective. A similar process was applied to the production of a lipid extract of Chlorella enriched with DHA, which contained 20% DHA (Sugimoto et al., 2002, Biol. Pharm. Bull 25:1090-1092). In this case, the percentage of EPA was slightly greater than 3%. This process, which is associated with the one already described for enrichment with vitamin B12, is the basis for the commercial product SUPER FRESH CHLORELLA SV-12 from Pacific Trading Co., Ltd.
The patent families that disclose the enrichment of Chlorella with polyunsaturated fatty acids, which in all cases use free fatty acids or the corresponding salts thereof, are:                KR2005015233-A; KR768757-B1: Process for producing Chlorella containing omega-3 fatty acids, including eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, which comprises the addition EPA and DHA monoglycerides to the culture medium at the end of the fermentation process.        JP10276684-A; KR98080312-A; JP3096654-B2; KR428732-B; KR423876-B: Production of Chlorella containing highly unsaturated fatty acids—it comprises culturing Chlorella in a medium with DHA and other highly unsaturated fatty acids in the form of free acids or the corresponding salts thereof.        
On the other hand, as far as the applicant is aware, there is only one reference that discloses the enrichment of a species of Nannochloropsis with DHA (Wacker et al., 2002, Limnol. Oceanogr. 47:1242-1248). In this reference, the freshwater species N. limnetica was enriched separately with pure EPA or DHA, or with a DHA-rich extract of the microalga Isochrysis aff. galbana (Clone T-ISO). This latter species contains high DHA values; however, they were very inefficiently transferred to N. limnetica, with a final ratio of 1 part of DHA for every 40 parts of EPA (weight:weight ratio) in the enriched biomass. Moreover, these authors clearly demonstrate the benefits of DHA enrichment in the diet of the mussel D. polymorpha, despite the low enrichment levels achieved using their methodology (Wacker et al., 2002, Limnol. Oceanogr. 47:1242-1248).
The crucial role of long-chain polyunsaturated fatty acids in the culture of various marine species has been extensively documented (Watanabe et al., 1983, Aquaculture 34:115-143; Izquierdo, 1996, Aquaculture Nutrition, 2: 183-191; Tocher, 2010, Aquaculture Research 41:717-732), although the presence of these fatty acids has also been identified as an essential factor in freshwater environments, which controls interactions in the nutritional chain (Müller-Navarra et al., 2000, Nature 403, 74-77).
In addition to applications in aquaculture, various species of the genus Nannochloropsis have been extensively studied as a source of EPA for nutritional applications in humans and animals (Sukenik, 1998, Cohen, Z. (Ed.), Chemicals from Microalgae. Taylor and Francis, London, p 41-56; Chini Zitelli et al., 1999, Journal of Biotechnology 70: 299-312), and, more recently, have received great attention due to their potential for biodiesel production (Rodolfi et al., 2008, Journal of Biotechnology 70: 299-312; Doan et al., 2011, Biomass and Bioenergy 35:2534-2544; San Pedro et al., 2013, Bioresource Technology 134:353-361).
In addition to applications in aquaculture, the different properties of EPA make it a compound of high biotechnological and pharmacological interest, hence the interest in using biomass from species of the genus Nannochloropsis, rich in this unsaturated fatty acid, in the field of human and animal nutrition. It has been demonstrated that the n-3 series polyunsaturated fatty acids EPA and DHA present a number of health benefits, and are effective in the treatment of cardiovascular diseases, including well-documented hypotriglycemic and anti-inflammatory effects. Similarly, several studies have suggested promising antihypertensive, anti-carcinogenic, anti-depression, anti-ageing and anti-arthritic effects. An anti-inflammatory and insulin-sensitising effect in metabolic disorders has also been disclosed. More specifically, various studies suggest that EPA may be beneficial in inflammatory processes, schizophrenia, depression, chronic fatigue syndrome, hepatic dysfunction, attention deficit hyperactivity syndrome, etc., and also improve the efficiency of chemotherapy in cancer processes (Siriwardhana et al., 2012, Se-Kwon Kim, (Eds.), Advances in Food and Nutrition Research, Academic Press, 2012, Volume 65, Pages 211-222). In animal experimentation systems, it has been demonstrated that the inclusion of EPA-rich Nannochloropsis biomass results in a greater proportion of DHA in the brain lipids of rat offspring, and also a better appearance and a greater DHA content in the eggs of hens fed with biomass from this species (Sukenik, 1999, Cohen, Z. (Ed.), Chemicals from Microalgae. Taylor and Francis, London, p 41-56).
If EPA-rich microalgal biomass is of great interest for application in the fields of aquaculture, animal breeding and the treatment of diseases in humans, arising from the aforementioned properties, obtaining biomass that is also enriched with DHA is of even greater interest. DHA is one of the main components of fish oil and, in addition to being essential for the development of marine species, it is very abundant in the brain phospholipids of mammals. It has been suggested that DHA is necessary for neuronal development and synaptic plasticity. The DHA content in brain phospholipids is also lower in patients with Alzheimer's disease. Moreover, the high DHA content in human breast milk has been related to the development of the central nervous system in children, which has led to the recommendation of supplementing formula milks with this compound. Other possible applications of DHA include anti-carcinogenic activity, psoriasis, etc. Moreover, numerous studies suggest that DHA is an important component for maintaining and improving cerebral functions in aged animals (Sugimoto et al., 2002, Biol. Pharm. Bull 25:1090-1092). Commercially, DHA designed for use in human nutrition is produced from fish oil or the heterotrophic dinoflagellate Crypthecodinium cohnii (Mendes et al., 2008, Journal of Applied Phycology 21:199-214). The use of DHA from C. cohnii for food enrichment, particularly in the field of aquaculture, is disclosed in the patent Gladue et al., 2002, U.S. Pat. No. 6,372,460 B1. Although there exist DHA-rich microalgae, with a DHA content that may range between 10% and 20% (Volkman et al., 1989, Journal of Experimental Marine Biology and Ecology, 128: 219-240), those species with a high content of this fatty acid present low levels of EPA.
Therefore, both in the field of aquaculture and in the field of animal and human nutrition, the availability of microalgal biomass simultaneously enriched with EPA and DHA is of great interest. A product with these characteristics has not been disclosed in the literature and is not in the market; consequently, it is still a challenge to obtain a microalga simultaneously enriched with EPA and DHA.